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1.
PLoS One ; 15(5): e0233017, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32407401

RESUMO

Myotonia congenita and hypokalemic periodic paralysis type 2 are both rare genetic channelopathies caused by mutations in the CLCN1 gene encoding voltage-gated chloride channel CLC-1 and the SCN4A gene encoding voltage-gated sodium channel Nav1.4. The patients with concomitant mutations in both genes manifested different unique symptoms from mutations in these genes separately. Here, we describe a patient with myotonia and periodic paralysis in a consanguineous marriage pedigree. By using whole-exome sequencing, a novel F306S variant in the CLCN1 gene and a known R222W mutation in the SCN4A gene were identified in the pedigree. Patch clamp analysis revealed that the F306S mutant reduced the opening probability of CLC-1 and chloride conductance. Our study expanded the CLCN1 mutation database. We emphasized the value of whole-exome sequencing for differential diagnosis in atypical myotonic patients.


Assuntos
Canais de Cloreto/genética , Paralisia Periódica Hipopotassêmica/complicações , Paralisia Periódica Hipopotassêmica/genética , Miotonia Congênita/complicações , Miotonia Congênita/genética , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Adolescente , Adulto , Idoso , Sequência de Aminoácidos , China , Canais de Cloreto/química , Canais de Cloreto/metabolismo , Consanguinidade , Sequência Conservada , Diagnóstico Diferencial , Feminino , Células HEK293 , Humanos , Paralisia Periódica Hipopotassêmica/metabolismo , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação , Miotonia Congênita/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Linhagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sequenciamento Completo do Exoma , Adulto Jovem
2.
Int J Mol Sci ; 21(7)2020 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-32276507

RESUMO

Myotonia congenita (MC) is a rare disorder characterized by stiffness and weakness of the limb and trunk muscles. Mutations in the SCN4A gene encoding the alpha-subunit of the voltage-gated sodium channel Nav1.4 have been reported to be responsible for sodium channel myotonia (SCM). The Nav1.4 channel is expressed in skeletal muscles, and its related channelopathies affect skeletal muscle excitability, which can manifest as SCM, paramyotonia and periodic paralysis. In this study, the missense mutation p.V445M was identified in two individual families with MC. To determine the functional consequences of having a mutated Nav1.4 channel, whole-cell patch-clamp recording of transfected Chinese hamster ovary cells was performed. Evaluation of the transient Na+ current found that a hyperpolarizing shift occurs at both the activation and inactivation curves with an increase of the window currents in the mutant channels. The Nav1.4 channel's co-expression with the Navß4 peptide can generate resurgent Na+ currents at repolarization following a depolarization. The magnitude of the resurgent currents is higher in the mutant than in the wild-type (WT) channel. Although the decay kinetics are comparable between the mutant and WT channels, the time to the peak of resurgent Na+ currents in the mutant channel is significantly protracted compared with that in the WT channel. These findings suggest that the p.V445M mutation in the Nav1.4 channel results in an increase of both sustained and resurgent Na+ currents, which may contribute to hyperexcitability with repetitive firing and is likely to facilitate recurrent myotonia in SCM patients.


Assuntos
Mutação de Sentido Incorreto , Miotonia Congênita/genética , Miotonia Congênita/fisiopatologia , Canal de Sódio Disparado por Voltagem NAV1.4/fisiologia , Sequência de Aminoácidos , Animais , Grupo com Ancestrais do Continente Asiático , Células CHO , Canalopatias/genética , Canalopatias/metabolismo , Canalopatias/fisiopatologia , Cricetulus , Feminino , Humanos , Masculino , Miotonia Congênita/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.4/química , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Técnicas de Patch-Clamp , Linhagem
3.
Muscle Nerve ; 61(6): 808-814, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32129495

RESUMO

INTRODUCTION: Mutations of the voltage-gated sodium channel gene (SCN4A), which encodes Nav1.4, cause nondystrophic myotonia that occasionally is associated with severe apnea and laryngospasm. There are case reports of nondystrophic myotonia due to mutations in the C-terminal tail (CTerm) of Nav1.4, but the functional analysis is scarce. METHODS: We present two families with nondystrophic myotonia harboring a novel heterozygous mutation (E1702del) and a known heterozygous mutation (E1702K). RESULTS: The proband with E1702K exhibited repeated rhabdomyolysis, and the daughter showed laryngospasm and cyanosis. Functional analysis of the two mutations as well as another known heterozygous mutation (T1700_E1703del), all located on EF hand-like motif in CTerm, was conducted with whole-cell recording of heterologously expressed channel. All mutations displayed impaired fast inactivation. DISCUSSION: The CTerm of Nav1.4 is vital for regulating fast inactivation. The study highlights the importance of accumulating pathological mutations of Nav1.4 and their functional analysis data.


Assuntos
Motivos EF Hand/genética , Potenciais da Membrana/fisiologia , Mutação/genética , Transtornos Miotônicos/diagnóstico , Transtornos Miotônicos/genética , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Pré-Escolar , Feminino , Células HEK293 , Humanos , Masculino , Pessoa de Meia-Idade , Transtornos Miotônicos/fisiopatologia , Adulto Jovem
4.
Biochim Biophys Acta Biomembr ; 1862(2): 183129, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31738900

RESUMO

Slow inactivation in voltage-gated Na+ channels (Navs) plays an important physiological role in excitable tissues (muscle, heart, nerves) and mutations that disrupt Nav slow inactivation can result in pathophysiologies (myotonia, arrhythmias, epilepsy). While the molecular mechanisms responsible for slow inactivation remain elusive, previous studies have suggested a role for the pore-lining D1-S6 helix. The goals of this research were to determine if (1) cysteine substitutions in D1-S6 affect gating kinetics and (2) methanethiosulfonate ethylammonium (MTSEA) accessibility changes in different kinetic states. Site-directed mutagenesis in the human skeletal muscle isoform hNav1.4 was used to substitute cysteine for eleven amino acids in D1-S6 from L433 to L443. Mutants were expressed in HEK cells and recorded from with whole-cell patch clamp. All mutations affected one or more baseline kinetics of the sodium channel, including activation, fast inactivation, and slow inactivation. Substitution of cysteine (for nonpolar residues) adjacent to polar residues destabilized slow inactivation in G434C, F436C, I439C, and L441C. Cysteine substitution without adjacent polar residues enhanced slow inactivation in L438C and N440C, and disrupted possible H-bonds involving Y437:D4 S4-S5 and N440:D4-S6. MTSEA exposure in closed, fast-inactivated, or slow-inactivated states in most mutants had little-to-no effect. In I439C, MTSEA application in closed, fast-inactivated, and slow-inactivated states produced irreversible reduction in current, suggesting I439C accessibility to MTSEA in all three kinetic states. D1-S6 is important for Nav gating kinetics, stability of slow-inactivated state, structural contacts, and state-dependent positioning. However, prominent reconfiguration of D1-S6 may not occur in slow inactivation.


Assuntos
Substituição de Aminoácidos , Cisteína/genética , Ativação do Canal Iônico , Canal de Sódio Disparado por Voltagem NAV1.4/química , Cisteína/química , Metanossulfonato de Etila/análogos & derivados , Metanossulfonato de Etila/química , Células HEK293 , Humanos , Simulação de Dinâmica Molecular , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Ligação Proteica , Domínios Proteicos
5.
J Neuromuscul Dis ; 6(4): 467-473, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31609695

RESUMO

BACKGROUND: The phenotypic spectrum of the skeletal muscle voltage-gated sodium channel gene (SCN4A) mutations has been expanding dramatically with advancements in genetic testing. Previously only known to cause autosomal dominant myotonia or periodic paralysis, now recessive mutations have been found causing congenital myopathies and congenital myasthenic syndromes. CASE PRESENTATION: A 27-year-old woman who was born with Arnold-Chiari malformation, hydrocephalus, high-arched palate, bilateral hip dysplasia, and severe scoliosis presented for evaluation of episodic muscle stiffness and weakness. Electrodiagnostic studies revealed myopathy and widespread myotonia. Muscle histopathology showed marked fiber size variability, type I fiber predominance with minimal scattered necrosis and regeneration which was typical of a congenital myopathy with an additional finding of a lobulated structural pattern in type I fibers. Sequential individual gene testing revealed a novel de novo heterozygous c.2386 C > G, p.Leu796Val missense mutation in the SCN4A gene. DISCUSSION: To the best of our knowledge, this is the first report of a dominant, heterozygous mutation in SCN4A causing a complex phenotype of congenital myopathy and myotonia with multiple congenital anomalies and unique muscle pathology findings. This case is another addition to the ever expanding phenotype of SCN4A mutations.


Assuntos
Doenças Musculares/genética , Mutação/genética , Miotonia Congênita/genética , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Adulto , Feminino , Testes Genéticos , Heterozigoto , Humanos , Músculo Esquelético/fisiopatologia , Miotonia Congênita/diagnóstico , Linhagem , Fenótipo
6.
Biomed Pharmacother ; 120: 109352, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31586905

RESUMO

Inflammatory monocyte and macrophage subset accumulation during the inflammatory response that drives atherosclerosis can exacerbate the extent of atherosclerosis. It has been demonstrated that voltage-gated sodium channels (VGSCs) can regulate cell bioactivities in monocytes/macrophages. We hypothesized that blockade of mononuclear phagocyte VGSCs was atheroprotective through monocyte/macrophage subset modulation and macrophage proliferation suppression in atherosclerotic lesions. In this experimental study, when VGSCs were knocked down with RNA interference plasmid transfection in mouse peripheral blood monocytes and monocyte-macrophage lineage RAW264.7 cells in vitro, the biological characteristics of proliferation, phagocytosis, and migration in RAW264.7 cells declined. In addition, suppression of LPS-induced M1 polarization and facilitation of IL-4-induced M2 polarization were also observed. In an in vivo study, ApoE knockout (ApoE-/-) mice were fed a standard chow diet (CD) or a western diet (WD). After feeding with phenytoin (PHT), no significant differences were detected in plasma lipids, and the anti-inflammatory phenotypes of both monocytes and macrophages were elevated and proinflammatory phenotypes declined. The local proliferation of macrophages was also distinctly suppressed, along with a significant reduction in atheromatous plaques. In conclusion, blockade of VGSCs in the mononuclear phagocyte system reduced atherosclerotic lesions, which may occur through altering monocyte/macrophage subsets and suppressing macrophage proliferation in atherosclerotic plaques. Blockage of VGSCs may play an important role in cardiovascular protection.


Assuntos
Aterosclerose/prevenção & controle , Ativação de Macrófagos , Macrófagos/metabolismo , Monócitos/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.9/metabolismo , Animais , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Proliferação de Células , Modelos Animais de Doenças , Regulação para Baixo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Monócitos/patologia , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Canal de Sódio Disparado por Voltagem NAV1.9/genética , Fagocitose , Placa Aterosclerótica , Células RAW 264.7 , Interferência de RNA , Transdução de Sinais
8.
J Genet ; 982019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31544778

RESUMO

Myotonia congenita (MC) is a Mendelian inherited genetic disease caused by the mutations in the CLCN1 gene, encoding the main skeletal muscle ion chloride channel (ClC-1). The clinical diagnosis of MC should be suspected in patients presenting myotonia, warm-up phenomenon, a characteristic electromyographic pattern, and/or family history. Here, we describe the largest cohort of MC Spanish patients including their relatives (up to 102 individuals). Genetic testing was performed by CLCN1 sequencing and multiplex ligation-dependent probe amplification (MLPA). Analysis of selected exons of the SCN4A gene, causing paramyotonia congenita, was also performed. Mutation spectrum and analysis of a likely founder effect of c.180+3A>T was achieved by haplotype analysis and association tests. Twenty-eight different pathogenic variants were found in the CLCN1 gene, of which 21 were known mutations and seven not described. Gross deletions/duplications were not detected. Four probands had a pathogenic variant in SCN4A. Two main haplotypes were detected in c.180+3A>T carriers and no statistically significant differences were detected between case and control groups regarding the type of haplotype and its frequencies. A diagnostic yield of 51% was achieved; of which 88% had pathogenic variants in CLCN1 and 12% in SCN4A. The existence of a c.180+3A>T founder effect remains unsolved.


Assuntos
Canais de Cloreto/genética , Miotonia Congênita/genética , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Estudos de Coortes , Éxons , Feminino , Efeito Fundador , Haplótipos , Humanos , Masculino , Músculo Esquelético/metabolismo , Mutação , Miotonia Congênita/diagnóstico , Polimorfismo de Nucleotídeo Único , Espanha
9.
Comput Biol Chem ; 83: 107132, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31563636

RESUMO

Here, we focused on exploring the selectivity mechanism against Nav1.7 over Nav1.4 due to different binding modes of two selected inhibitors. By the superposition of Nav1.7 and Nav1.4 proteins, we selected the most homologous chain of Nav1.7 with Nav1.4, defining the active site of Nav1.4-VSD4 based on the aryl sulfonamide binding site of Nav1.7-VSD4. Comparison of the conformations exhibited by Tyr1386 (Nav1.4) and Tyr1537 (Nav1.7) suggested that the steric hindrance caused by Tyr1386 owned primary influence on inhibition selectivity, which was further verified through molecular docking and MD simulation of two representative inhibitors. Our finding would be helpful for discovery of selective Nav1.7 inhibitors over Nav1.4.


Assuntos
Modelos Moleculares , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Sulfonamidas/farmacologia , Sítios de Ligação/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Ligantes , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/química , Software , Relação Estrutura-Atividade , Sulfonamidas/química , Termodinâmica
10.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 36(8): 809-812, 2019 Aug 10.
Artigo em Chinês | MEDLINE | ID: mdl-31400134

RESUMO

OBJECTIVE: To explore the clinical features of a Chinese pedigree affected with skeletal muscle sodium channelopathies due to variation of SCN4A gene. METHODS: Potential variation of the 24 exons of the SCN4A gene was screened using PCR and Sanger sequencing. RESULTS: Four family members were affected with the disease in an autosomal dominant inheritance pattern. Three patients had normekalemic periodic paralysis, while 1 showed paramyotonia congenita. Genetic analysis detected a missense variation c.2078T>C (p.Ile693Thr) in exon 13 of the SCN4A gene in the proband and other 3 affected relatives. CONCLUSION: Normokalemic periodic paralysis and paramyotonia congenita can occur in different family members with skeletal muscle sodium channelopathies due to c.2078T>C(p.Ile693Thr) variation of SCN4A gene.


Assuntos
Canalopatias/genética , Músculo Esquelético/fisiopatologia , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Humanos , Mutação , Linhagem
11.
J Biomol NMR ; 73(10-11): 531-544, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31281943

RESUMO

Assignment of backbone resonances is a necessary initial step in every protein NMR investigation. Standard assignment procedure is based on the set of 3D triple-resonance (1H-13C-15N) spectra and requires at least several days of experimental measurements. This limits its application to the proteins with low stability. To speed up the assignment procedure, combinatorial selective labeling (CSL) can be used. In this case, sequence-specific information is extracted from 2D spectra measured for several selectively 13C,15N-labeled samples, produced in accordance with a special CSL scheme. Here we review previous applications of the CSL approach and present novel deterministic 'CombLabel' algorithm, which generates CSL schemes minimizing the number of labeled samples and their price and maximizing assignment information that can be obtained for a given protein sequence. Theoretical calculations revealed that CombLabel software outperformed previously proposed stochastic algorithms. Current implementation of CombLabel robustly calculates CSL schemes containing up to six samples, which is sufficient for moderately sized (up to 200 residues) proteins. As a proof of concept, we calculated CSL scheme for the first voltage-sensing domain of human Nav1.4 channel, a 134 residue four helical transmembrane protein having extremely low stability in micellar solution (half-life ~ 24 h at 45 °C). Application of CSL doubled the extent of backbone resonance assignment, initially obtained by conventional approach. The obtained assignment coverage (~ 50%) is sufficient for ligand screening and mapping of binding interfaces.


Assuntos
Sequência de Aminoácidos , Proteínas de Membrana/química , Ressonância Magnética Nuclear Biomolecular/métodos , Humanos , Canal de Sódio Disparado por Voltagem NAV1.4/química , Estudo de Prova de Conceito , Ligação Proteica , Domínios Proteicos , Software , Coloração e Rotulagem , Fatores de Tempo
12.
Mar Drugs ; 17(7)2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31269696

RESUMO

Cyclic µ-conotoxin PIIIA, a potent blocker of skeletal muscle voltage-gated sodium channel NaV1.4, is a 22mer peptide stabilized by three disulfide bonds. Combining electrophysiological measurements with molecular docking and dynamic simulations based on NMR solution structures, we investigated the 15 possible 3-disulfide-bonded isomers of µ-PIIIA to relate their blocking activity at NaV1.4 to their disulfide connectivity. In addition, three µ-PIIIA mutants derived from the native disulfide isomer, in which one of the disulfide bonds was omitted (C4-16, C5-C21, C11-C22), were generated using a targeted protecting group strategy and tested using the aforementioned methods. The 3-disulfide-bonded isomers had a range of different conformational stabilities, with highly unstructured, flexible conformations with low or no channel-blocking activity, while more constrained molecules preserved 30% to 50% of the native isomer's activity. This emphasizes the importance and direct link between correct fold and function. The elimination of one disulfide bond resulted in a significant loss of blocking activity at NaV1.4, highlighting the importance of the 3-disulfide-bonded architecture for µ-PIIIA. µ-PIIIA bioactivity is governed by a subtle interplay between an optimally folded structure resulting from a specific disulfide connectivity and the electrostatic potential of the conformational ensemble.


Assuntos
Conotoxinas/farmacocinética , Canal de Sódio Disparado por Voltagem NAV1.4/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Conotoxinas/química , Dissulfetos/química , Isomerismo , Simulação de Acoplamento Molecular , Conformação Proteica , Eletricidade Estática , Relação Estrutura-Atividade , Bloqueadores do Canal de Sódio Disparado por Voltagem/química
15.
BMC Neurol ; 19(1): 125, 2019 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-31189464

RESUMO

BACKGROUND: Non-dystrophic myotonias (NDMs) are skeletal muscle disorders involving myotonia distinct from myotonic dystrophy. It has been reported that the muscle pathology is usually normal or comprises mild myopathic changes in NDMs. We describe various pathological findings mimicking those of myotonic dystrophy (DM) in biopsied muscle specimens from a patient with NDMs with a long disease duration. CASE PRESENTATION: A 66-year-old Japanease man presented eye closure myotonia, percussion myotonia and grip myotonia together with the warm-up phenomenon and cold aggravation from early childhood. On genetic analysis, a heterozygous mutation of the SCN4A gene (c.2065 C > T, p.L689F), with no mutation of the CLCN1, DMPK, or ZNF9/CNBP gene, was detected. He was diagnosed as having NDMs. A biopsy of the biceps brachii muscle showed increasing fiber size variation, internal nuclei, chained nuclei, necrotic fibers, fiber splitting, endomysial fibrosis, pyknotic nuclear clumps and disorganized intermyofibrillar networks. Sarcoplasmic masses, tubular aggregates and ragged-red fibers were absent. CONCLUSION: It is noteworthy that the present study revealed various pathological findings resembling those seen in DM, although the pathology is usually normal or mild in NDMs. The pathological similarities may be due to muscular modification with long-standing myotonia or excessive muscle contraction based on abnormal channel activity.


Assuntos
Músculo Esquelético/patologia , Miotonia/genética , Miotonia/patologia , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Idoso , Heterozigoto , Humanos , Masculino , Mutação
16.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 48(1): 12-18, 2019 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-31102352

RESUMO

OBJECTIVE: To investigate the mechanism of congenital paramyotonia caused by human skeletal muscle voltage-gated sodium channel hNav1.4 mutant I1363T. METHODS: The conservation of the mutant site were detecled by using amino acid sequence alignment; the C-terminal mCherry fusion hNav1.4 was constructed, and the expression and distribution of wild type and hNav1.4 mutant I1363T were determined by confocal microscopy; the steady-state activation, fast inactivation and window current of wild type and hNav1.4 mutant I1363T were examined by whole-cell patch clamp. RESULTS: Alignment of the amino acid sequences revealed that Ile1363 is highly conserved in human sodium channels. There was no significant difference in expression level and distribution between wild type and I1363T. Although no significant differences were observed between I1363T mutant and wild type in the activation upon channel gating, the V0.5 of voltage-dependence of fast inactivation of I1363T mutant[(-59.01±0.26) mV] shifted 9 mV towards depolarization as compared with wild type[(-68.03±0.34) mV], and the slope factor of voltage-dependence curve increased to (5.24±0.23) mV, compared with (4.55±0.21) mV of the wild type. Moreover, I1363T showed the larger window current than that of the wild type. CONCLUSIONS: I1363T causes the defect in fast inactivation of hNav1.4, which may increase the excitability of muscle cells and be responsible for myotonia. The increased window current of I1363T may result in an increase of inward Na+ current, could subsequently inactivate the channels and lead to loss of excitability and paralysis.


Assuntos
Músculo Esquelético , Canal de Sódio Disparado por Voltagem NAV1.4 , Perfilação da Expressão Gênica , Humanos , Ativação do Canal Iônico/genética , Músculo Esquelético/fisiopatologia , Mutação , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Análise de Sequência de Proteína
17.
Channels (Austin) ; 13(1): 110-119, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-30931713

RESUMO

OBJECTIVE: To verify the diagnosis of channelopathies in two families and explore the mechanism of the overlap between periodic paralysis (PP) and paramyotonia congenita (PMC). METHODS: We have studied two cases with overlapping symptoms of episodic weakness and stiffness in our clinical center using a series of assessment including detailed medical history, careful physical examination, laboratory analyses, muscle biopsy, electrophysiological evaluation, and genetic analysis. RESULTS: The first proband and part of his family with the overlap of PMC and hyperkalemic periodic paralysis (HyperPP) has been identified as c.2111C > T (T704M) substitution of the gene SCN4A. The second proband and part of his family with the overlap of PMC and hypokalemic periodic paralysis type 2 (HypoPP2) has been identified as c.4343G > A (R1448H) substitution of the gene SCN4A. In addition, one member of the second family with overlapping symptoms has been identified as a novel mutation c.2111C > T without the mutation c.4343G > A. CONCLUSIONS: SCN4A gene mutations can cause the overlap of PMC and PP (especially the HypoPP2). The clinical symptoms of episodic weakness and stiffness could happen at a different time or temperature. Based on diagnosis assessments such as medical history and muscle biopsy, further evaluations on long-time exercise test, genetic analysis, and patch clamp electrophysiology test need to be done in order to verify the specific subtype of channelopathies. Furthermore, the improvement of one member in the pregnancy period can be used as a reference for the other female in the child-bearing period with T704M.


Assuntos
Transtornos Miotônicos/genética , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Paralisia Periódica Hiperpotassêmica/genética , Adolescente , Adulto , Humanos , Masculino , Mutação , Transtornos Miotônicos/patologia , Paralisia Periódica Hiperpotassêmica/patologia , Linhagem , Adulto Jovem
18.
Nat Commun ; 10(1): 1514, 2019 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-30944319

RESUMO

Skeletal muscle voltage-gated Na+ channel (NaV1.4) activity is subject to calmodulin (CaM) mediated Ca2+-dependent inactivation; no such inactivation is observed in the cardiac Na+ channel (NaV1.5). Taken together, the crystal structures of the NaV1.4 C-terminal domain relevant complexes and thermodynamic binding data presented here provide a rationale for this isoform difference. A Ca2+-dependent CaM N-lobe binding site previously identified in NaV1.5 is not present in NaV1.4 allowing the N-lobe to signal other regions of the NaV1.4 channel. Consistent with this mechanism, removing this binding site in NaV1.5 unveils robust Ca2+-dependent inactivation in the previously insensitive isoform. These findings suggest that Ca2+-dependent inactivation is effected by CaM's N-lobe binding outside the NaV C-terminal while CaM's C-lobe remains bound to the NaV C-terminal. As the N-lobe binding motif of NaV1.5 is a mutational hotspot for inherited arrhythmias, the contributions of mutation-induced changes in CDI to arrhythmia generation is an intriguing possibility.


Assuntos
Cálcio/metabolismo , Calmodulina/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Sítios de Ligação , Cálcio/química , Calmodulina/química , Calmodulina/genética , Humanos , Modelos Moleculares , Músculo Esquelético/metabolismo , Mutação , Canal de Sódio Disparado por Voltagem NAV1.4/química , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Canal de Sódio Disparado por Voltagem NAV1.5/química , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas
19.
Dokl Biochem Biophys ; 484(1): 9-12, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31012002

RESUMO

An effective bacterial system for the production of ß-toxin Ts1, the main component of the Brazilian scorpion Tityus serrulatus venom, was developed. Recombinant toxin and its 15N-labeled analogue were obtained via direct expression of synthetic gene in Escherichia coli with subsequent folding from the inclusion bodies. According to NMR spectroscopy data, the recombinant toxin is structured in an aqueous solution and contains a significant fraction of ß-structure. The formation of a stable disulfide-bond isomer of Ts1, having a disordered structure, has also been observed during folding. Recombinant Ts1 blocks Na+ current through NaV1.5 channels without affecting the processes of activation and inactivation. At the same time, the effect upon NaV1.4 channels is associated with a shift of the activation curve towards more negative membrane potentials.


Assuntos
Venenos de Escorpião , Bloqueadores dos Canais de Sódio , Animais , Humanos , Proteínas Musculares/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Venenos de Escorpião/biossíntese , Venenos de Escorpião/química , Venenos de Escorpião/isolamento & purificação , Venenos de Escorpião/farmacologia , Bloqueadores dos Canais de Sódio/química , Bloqueadores dos Canais de Sódio/isolamento & purificação , Bloqueadores dos Canais de Sódio/farmacologia , Canais de Sódio/metabolismo , Relação Estrutura-Atividade , Xenopus laevis
20.
Neurology ; 92(13): e1405-e1415, 2019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-30824560

RESUMO

OBJECTIVE: To identify the genetic and physiologic basis for recessive myasthenic congenital myopathy in 2 families, suggestive of a channelopathy involving the sodium channel gene, SCN4A. METHODS: A combination of whole exome sequencing and targeted mutation analysis, followed by voltage-clamp studies of mutant sodium channels expressed in fibroblasts (HEK cells) and Xenopus oocytes. RESULTS: Missense mutations of the same residue in the skeletal muscle sodium channel, R1460 of NaV1.4, were identified in a family and a single patient of Finnish origin (p.R1460Q) and a proband in the United States (p.R1460W). Congenital hypotonia, breathing difficulties, bulbar weakness, and fatigability had recessive inheritance (homozygous p.R1460W or compound heterozygous p.R1460Q and p.R1059X), whereas carriers were either asymptomatic (p.R1460W) or had myotonia (p.R1460Q). Sodium currents conducted by mutant channels showed unusual mixed defects with both loss-of-function (reduced amplitude, hyperpolarized shift of inactivation) and gain-of-function (slower entry and faster recovery from inactivation) changes. CONCLUSIONS: Novel mutations in families with myasthenic congenital myopathy have been identified at p.R1460 of the sodium channel. Recessive inheritance, with experimentally established loss-of-function, is a consistent feature of sodium channel based myasthenia, whereas the mixed gain of function for p.R1460 may also cause susceptibility to myotonia.


Assuntos
Síndromes Miastênicas Congênitas/genética , Canal de Sódio Disparado por Voltagem NAV1.4/genética , Adulto , Animais , Eletromiografia , Feminino , Finlândia , Humanos , Laringismo/genética , Laringismo/fisiopatologia , Mutação com Perda de Função , Masculino , Hipotonia Muscular/genética , Hipotonia Muscular/fisiopatologia , Músculo Esquelético/patologia , Mutação de Sentido Incorreto , Síndromes Miastênicas Congênitas/metabolismo , Síndromes Miastênicas Congênitas/fisiopatologia , Miotonia/genética , Miotonia/fisiopatologia , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Oócitos , Técnicas de Patch-Clamp , Linhagem , Sequenciamento Completo do Exoma , Xenopus
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